1. Field of the Invention
The field of this invention is the production of recombinant procollagen in yeast.
2. Background
Collagen has provided a number of commercial applications, including use as a convenient substrate for cell culture, as well as in the manufacture of biocompatible materials (e.g. artificial skin) having a variety of therapeutic applications in humans. Commercially available collagens are primarily isolated from freshly killed farm animals such as cows and pigs in polymerized form or in soluble form derived by enzymatic or chemical treatment of polymerized collagen. However, polymerized collagens and soluble collagens derived from polymerized collagens are of limited usefulness due to the presence of natural crosslinks within the collagen. Furthermore, non-human collagen can provoke undesirable immune responses when administered to human subjects.
As one alternative, human collagen can be purified from human sources such as human placenta, as described in U.S. Pat. No. 5,002,071 (Research Development Foundation) and in copending U.S. application Ser. No. 07/921,810 (Collagen Corporation). In addition to source limitations and the risk of contamination by human pathogens such as Hepatitis viruses and HIV, the methods for recovering collagen from these sources bias the collagen type recovered. Furthermore, because the collagen is derived from naturally crosslinked tissues, the collagen recovered is not entirely homogeneous. Another approach is the expression of recombinant human collagen in the milk of transgenic animals, as described in copending U.S. applications Ser. Nos. 08/183,648 and 08/011,643 (Collagen Corporation). However, this approach subjects the recombinant collagen to any host deficiencies in translational processing.
The term collagen refers to a type of protein that encompasses a class of structurally-related polypeptides consisting of helical collagen chains and homo and heteromeric polymers thereof. Collagen chains often contain both long helical domains and non-helical extensions, or telopeptides. The helical domains comprise xe2x80x94(GXY)nxe2x80x94 repeats, where X and/or Y are frequently proline or hydroxyproline.
Collagen chains are encoded by a number of related genes, See, Adams, S. L. Amer J Respir Cell and Mol Biol (1989) 1:161-168; Mayne and Brewton (1993) Current Opinion in Cell Biology 5:883-890; van der Rest and Bruckner (1993) Current Opinion in Structural Biology 3:430-436. Collagen-encoding transcripts are initially translated into procollagen chains which undergo a variety of post-translational events such as processing, secretion and assembly (e.g. disulfide exchange, lysyl residue hydroxylation, glycosylation, and crosslinking of the helical chains, see for example, Prockop et al. (1984) New England J Med 3111:376-386) to form mature collagen chain helices. In addition, procollagens contain N- and C-terminal propeptides that facilitate collagen formation, See, e.g. Lee et al. (1992) J Biol Chem 267, 24126-24133.
It is these unique properties of collagen, including the protein size and structure, the requisite postranslational processing, the secretory pathway, the nature of the collagen transcript and gene itself and its assembly into trimeric molecules, etc., that has severely restricted the ability of in vitro cellular expression systems to produce collagen or procollagen.
Relevant Literature
For reviews, see Nothwehr et al. (1994) J. Biol. Chem 269,10185-10188 re the sorting of membrane proteins in the yeast secretory pathway; Gellissen et al. (1992) van Leeuwenhoek 62, 79-93 re heterologous protein production in yeast; and, Hitzeman et al. (1990) Meth Enzymology 185, 421-441 re the use of signal sequences for secretion of heterologous proteins from yeast. Robinson et al. (1994) Biotechnology 12, 381-384 describes the use of protein disulfide isomerase overexpression to increase secretion of foreign proteins in yeast. Prockop et al. (1997) U.S Pat. No. 5,593,859 relates to synthesis of collagens in recombinant systems and Vuorela et al. (1997) EMBO J. 16, 6702-6712 reportedly expressed collagen proteins in Pichia pastoris. 
The invention provides methods and compositions for making a mammalian procollagen in yeast. Generally, the subject methods involve (a) incubating a recombinant yeast cell comprising: (i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter; (ii) a prolyl hydroxylase gene; (iii) a protein disulfide isomerase gene, wherein the prolyl hydroxylase and protein disulfide isomerase genes are expressed to produce polypeptides that are associated into active proteins during the incubating step, in a medium under conditions wherein the collagen genes are expressed as procollagen chains and the chains secreted in the form of triple helical procollagen; and (b) recovering the triple helical procollagen. In various particular embodiments, the first single divergent heterologous promoter is a GAL1-10 promoter, the collagen genes reside on a plasmid or multiple integrants in the cell, the prolyl hydroxylase and protein disulfide isomerase genes are integrated into the genome of the yeast, the prolyl hydroxylase gene and protein disulfide isomerase genes are operably linked to a second single divergent heterologous promoter, the first and second single divergent heterologous promoters are the same, the protein disulfide isomerase gene comprises a signal sequence heterologous to the protein disulfide isomerase gene, the collagen and hydroxylase genes each comprise homologous secretion signal sequences, the medium comprises supplemental casein amino acids sufficient to promote the hydroxylation of the collagen under the conditions, the yeast cell is a Saccharomyces cerevisiae and the mammalian collagen genes are human collagen genes. The subject compositions include recombinant yeast cells useful for making a mammalian procollagen. Such cells generally comprise: (i) a stable genetic construct comprising in opposite orientations, first and second mammalian collagen genes operably linked to a first single divergent heterologous promoter; (ii) a prolyl hydroxylase gene; and (iii) a protein disulfide isomerase gene.